Objective: To investigate the mechanism of Hul5 noncatalytic activity and its functional coupling to Ubp6 in protein degradation at the proteasome. Hypothesis: I hypothesize that there is a discrete noncatalytic function of Hul5 and that it inhibits the deubiquitinating activity of UbpG to ensure commitment to and accelerate substrate degradation. Specific Aims: (1) To characterize the functional regions of HulS. (2) To investigate the link between Ubp6 and the noncatalytic function of Hul5. (3) To examine the in vivo dependence of substrate degradation by the proteasome on the noncatalytic activity of Hul5. Research design: The model organism utilized in these studies will be S. cerevisiae. The initial studies will be focused on identifying the specific regions of Hul5 that are responsible for its many functions. Truncations and mutations in Hul5 will be generated and screened for phenotypes in yeast. Mutants will be isolated that are deficient in noncatalytic function, proteasome docking, and ubiquitin chain binding, respectively. Next, the mutants generated in Hul5 will be combined with mutants in Ubp6 to elucidate the interplay between Hul5 and Ubp6 through phenotypic screening. Following this, the functional coupling of Hul5 and Ubp6 will be reconstituted in vitro by altering the composition of the proteasome and measuring degradation of a characterized substrate. Finally, mass spectrometry will be utlized to identify endogenous substrates affected by Hul5 noncatalytic activity. Detailed analysis of the degradation rates of these identified substrates will reveal the importance and contribution of Hul5 to global protein degradation. In addition, combinations of Hul5 and Ubp6 mutants will be analyzed by mass spectrometry to investigate how the coupling of their antagonizing functions controls proteasome function and how imbalance in Hul5 and Ubp6 activity can affect substrate degradation. Relevance to public health: This proposal aims to investigate the fundamentals of the commitment of substrates to proteasomal degradation. The improper disposal of proteins that regulate cell growth and division is a well documented instigant of cancer. A large number of proteasome inhibitors are currently in clinical trials for cancer treatment. Therefore, a better understanding of the regulation of the proteasome will aid in the development of future approaches to cancer prevention and treatment.